|Ahead of print publication
Comparative scores of ABO and Rh hemagglutination reactions
Prashant Devidas Khuje1, Mahesh S Karandikar1, Bharti U Sable2
1 Department of Physiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pimpri, Pune, India
2 Sable Pathology Laboratory, Warora, Chandrapur, Maharashtra, India
|Date of Submission||16-Oct-2020|
|Date of Decision||16-Oct-2020|
|Date of Acceptance||19-Jun-2021|
Prashant Devidas Khuje,
Department of Physiology, DR.D.Y.P.M.C, Pimpri, Pune, 411018, F-104, Wing-H, Eden-II, Mahindra Royale, CTS 6020, Pimpri, Pune - 411 018, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Blood group determination is done by the slide test and tube test. Grading/scoring of hemagglutination reactions is performed through the naked eye and microscopy to detect spurious (false positive and negative) reactions. Objectives: In our observational study, comparative scoring of ABO and Rh hem-agglutination reactions was done based on degree of hemolysis and agglutination through the macroscopic and microscopic technique. Materials and Methods: The determination of blood group of 209 participants was done by the porcelain slide method using commercially prepared antisera (manufactured by TULIP DIAGNOSTICS (P) LTD.) containing anti-A, anti-B, and anti-D monoclonal antibodies followed by scoring/grading based on degree of hemolysis and red blood cell clumping. The data were statistically analyzed using the Chi-square test and Z-test of proportion. Results: ABO hemagglutination reactions showed significantly higher score ([A-Macro Z = 7.153, Micro Z = 4.65] [B-Macro Z = 11.17, Micro Z = 21]) (Z >2.5, P < 0.001) than Rh hemagglutination reactions. B hemagglutination reactions showed insignificantly higher score (B-Macro Z = 1.29, Micro Z = 1.816) (Z <1.95, P < 0.05) than A hemagglutination reaction. Conclusion: ABO hemagglutination reactions showed significantly higher score than Rh hemagglutination reactions as ABO system agglutinin are pentavalent (immunoglobulin M) binding 10 agglutinogens while that of Rh system are bivalent binding 4 agglutinogens. B hemagglutination reactions showed insignificantly higher score than A hem-agglutination reaction which required further evaluation.
Keywords: ABO RH scores
| Introduction|| |
Red blood cell (RBC) surface membrane contains a variety of genetically determined blood group agglutinogens (BGs antigens), while plasma contains noncorrespondent agglutinins (antibody).,, However, the ABO and Rh BG system has fundamental importance in transfusions. In clinical laboratories, it is the standard procedure to test for BGs A (containing only A antigens), B (containing only B antigens), AB (having both A- and B antigens), O (neither A nor B antigens), and Rh (giving information about the presence or absence of Rh antigens).,, During blood group determination, the red cells having surface agglutinogens are made to react with the commercially available antisera-containing known agglutinins. The presence or absence of clumping (agglutination) and hemolysis of red cells determines the blood group according to the ABO and Rh system. Grading/scoring of agglutination reactions is performed using the slide or tube sample.,
Scoring assists in detecting the presence of more than one antibody, dosage effect of antigens, abnormal weak reactions, and spurious (false positive) reactions.
The present study was planned to examine the scores of ABO and Rh blood group hem-agglutination reactions by the naked eye (NE) examination and simple microscopy according to the degree of hemolysis and agglutination followed by comparing the ABO and Rh system hemagglutination reactions.
| Materials and Methods|| |
After receiving approval from the Institutional Ethics Committee, determination of blood group of 209 participants was done by the porcelain slide method followed by scoring after getting participants' informed consent.
Fresh capillary blood samples from normal healthy male and female adults of age ranged between 18 and 25 years were included in the study.
Stored blood, clotted blood samples, and blood samples with any in vitro anticoagulants and unwilling participants were excluded from the study.
Six pits arranged in two rows of three each on a dry and clean porcelain tile were labeled as A (ABO antigen A), C (control) and B (ABO antigen B) on top row, and D (Rh antigen D) and S (red cell suspension [RCS]) on the 1st and 2nd pits of the second row, respectively.
A drop of commercially prepared antisera (manufactured by TULIP DIAGNOSTICS (P) LTD.) containing monoclonal Anti-A, Anti-B, and Anti-D antibodies (directed against corresponding blood group antigens) and citrated normal saline were poured in pits labeled A, B, D, and C pits, respectively, with separate droppers for each fluid. After pricking the finger under all aseptic precautions, two drops of blood were poured and mixed with 4 drops of citrated normal saline contained in pit “S” to get a homogeneous RCS.,,,
A drop of this suspension was transferred to each of the pit A, C, B, and D. Mixing of the contents was done with separate glass rod for each pit solutions.
The reactions were read by single and same observer after 6–10 min with intermittent blowing at 37°C., Pit C acted as the control to check contamination.
The nature and type of blood group antigen and antibody decide the formation of agglutinate and degree of hemolysis. ABO and Rh agglutination reactions were then scored by NE, as follows after blowing the solution in the pit for 5 s.
Grade 4: – single solid agglutinate with/without clear background
Grade 3:– 2 to 4 large agglutinates with/without clear background
Grade 2:– 5 to10 medium-sized agglutinates with/without clear background
Grade 1:– >10 small aggregates with/without clear background
Grade 0:– Granular turbid suspension.
After NE examination, scoring by microscopy (M) was done by same observer through wet slide preparation of the pit solution placed on the glass slide followed by placing a cover slip and was then observed and interpreted by same observer under low power magnification.,
Grade 4: – Single large aggregate per field with/without free RBCs
Grade 3: – 2 to 4 large aggregates per field with/without free RBCs
Grade 2: – 5 to 10 medium-sized aggregates per field with/without free RBCs
Grade1: – >10 small aggregates per field in the background of RBCs
Grade 0: – No aggregates in the background of free RBCs
Negative: – Freely scattered RBCs with no aggregates.
The data were statistically analyzed by the Chi-square test and Z-test of proportion.
| Results|| |
ABO hemagglutination reactions showed significantly higher score ([A-Macro Z = 7.153, Micro Z = 4.65] [B-Macro Z = 11.17, Micro Z = 21]) (Z > 2.5, P < 0.001) than Rh hemagglutination reactions. B hemagglutination reactions showed insignificantly higher score (B-Macro Z = 1.29, Micro Z = 1.816) (Z <1.95, P < 0.05) than A hemagglutination reaction [Table 1]. Eleven negative reactions detected by macroscopic examination were confirmed as true positive by microscopy.
| Discussion|| |
In the present study, porcelain slide was used for blood group typing and macroscopic scoring followed by microscopy. Plain slides were for blood group determination.,
Hemagglutination reactions were graded using the slide samples. They were graded as single large aggregate (Grade 4+), 2–3 large to medium aggregates (Grade 3+), 4–7 medium to small masses (Grade 2+), and many small aggregates in the background of free red cells (Grade 1+).,
Grading of ABO and Rh agglutination reactions was done macroscopically using the test tube samples. NE examinations of reactions within the tubes after shaking described the grades as Grade (4+) with 1 big clump, Grade (3+) with 2 or 3 clumps, Grade (2+) with many small clumps with clear supernatant, Grade (1+) with many small clumps with turbid supernatant and Grade (Zero or H) with smooth suspension.,
Hemagglutination reactions interpretation was done based on microscopic examination using centrifuged test tube blood sample. These grades were described as Grade (4+): solid agglutinate with clear background, Grade (3+): with breaking of solid agglutinate into several large agglutinates with clear background, Grade (2+) with many medium-sized agglutinates with clear background but no free RBCs, Grade (1+) with many small aggregates/clumps barely visible macroscopically with a turbid background and many free RBCs, and Grade (0) or negative with no clumps with free RBCs.,
In the present study, comparison between ABO and Rh system hemagglutination reactions was done along with intra-ABO hemagglutination (A and B) reaction. ABO hemagglutination reactions showed significantly higher score than Rh hemagglutination reactions. B hemagglutination reactions showed insignificantly higher score than A hemagglutination reaction.
ABO group system agglutinin is pentavalent immunoglobulin M (IgM) binding 10 agglutinogens (forming comparatively larger agglutinates) while that of Rh agglutinin are bivalent-binding four agglutinogens. Therefore, ABO blood group system agglutinates showed higher scores than Rh blood group system agglutinate. B hemagglutination reactions showed higher scores than A.
| Conclusion|| |
ABO hemagglutination reactions showed significantly higher score than Rh hemagglutination reactions as ABO agglutinin are pentavalent (IgM) binding 10 agglutinogens while that of Rh system are bivalent-binding four agglutinogens. B hemagglutination reactions showed insignificantly higher score than A hemagglutination reaction which require further evaluation.
Modern-day methods/techniques such as microplate, column gel configuration, nucleic acid amplification, etc., should have been done to determine the specific BGs antigens and to quantify hemagglutination reactions.
Financial support and sponsorship
This study was financially supported by Dr. D. Y. Patil Medical College, Hospital and Research Center, Pimpri, Pune - 411 018, Maharashtra, India.
Conflicts of interest
There are no conflicts of interest.
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